三苯基甲烷类染料氧化酶基因(tpmD)在大肠杆菌中的无诱导表达

为了解决嗜水气单胞菌DN322对鱼类的潜在致病性问题,有必要克隆表达该菌的三苯基甲烷染料脱色酶基因tpmD.通过PCR方法获得该基因,并与脱色希瓦氏菌S12的NAD(P)H脱氢酶基因启动子和用于荧光标记的编码CCPGCC的碱基序列融合,将有启动子和无启动子的融合基因片段分别连接到质粒pMD18-T中,转化大肠杆菌E.coli DH5α和E.coli BL21.结果发现,有启动子的融合基因能被E.coil在不加诱导物IPTG的条件下高效正确表达,脱色酶活性甚至超过基因供体菌DN322.将上述两株大肠杆菌工程菌株在自然水体中进行孔雀石绿污染小试处理,60d内在每个350mL反应体系中累积共投加大于10000mg的孔雀石绿,脱色率一直保持在92%以上;细菌数量最后增加了5～10倍.研究结果证明,基因工程菌在不加营养物的条件下也有很强的脱色活性和长期的存活能力;在tpmD基因3'端加入的用于螯合双砷荧光染料的18bp编码氨基酸碱基序列不影响此基因的表达和酶的脱色活性,这将赋予此工程菌可示踪性.     

高效脱色菌群的脱色、产酶及群落分析

富集筛选到2组能够在开放条件下对多种染料和造纸废水高效脱色的混合菌群,以该混合菌群构建的生物膜反应器在连续运行条件下,对COD为750～1175mg/L、色度为200～320的染料废水进行脱色、降解,脱色率为80%～90%、COD去除率为69%～90%.该混合菌群能够在不同染料培养基中、造纸废水或染料废水中产生木质素降解酶系统中的1种或2种酶.分析表明,2组菌群中真菌与细菌的比例为6.8:1～51.8:1,优势微生物种类为真菌,从中分离出16株真菌,其中8株对活性红M-3BE具有明显脱色效果.     

红芝所产漆酶对蒽醌染料的脱色研究

以红芝Ganoderma lucidum发酵所得漆酶粗酶液对蒽醌染料进行脱色实验,分别考察了反应时间、染料浓度、温度、pH、转速等条件对蒽醌染料脱色的影响。结果表明,红芝漆酶粗酶液对蒽醌染料具有很好的脱色效果,脱色的适宜条件为:染料浓度150mg/L、反应温度40℃、pH值3.5。在上述条件下反应8h,蒽醌染料脱色率可达90%左右,摇床转速对染料脱色影响不大。     

基因工程菌漆酶对蒽醌染料的脱色研究

利用基因工程菌甲醇毕赤酵母(PMAD16/pMETαA-Lcc1)培养产生的重组漆酶,研究了重组漆酶对蒽醌染料Remazol Brilliant Blue R脱色的影响。结果表明:重组漆酶对蒽醌染料RBBR脱色的最佳pH值为4.5,温度为45℃,当溶液中漆酶活力为10.0U/mL时,在最适脱色条件下作用14h,粗漆酶和纯化漆酶对蒽醌染料RBBR(80mg/L)的脱色率分别为95.2%和87.5%,重组漆酶可有效地使蒽醌染料RBBR脱色。     

漆酶催化复合染料酸性黑ATT脱色的研究

利用白腐菌Ganoderma lucidum wz-32发酵制备的漆酶对印染工业常用的复合染料酸性黑ATT进行催化脱色.在单因子试验的基础上,通过正交优化试验确定酸性黑ATT的最佳脱色条件为:温度50℃,染料浓度0.1 g·L-1,酶量9 U·mL-1,pH 5.0,最适条件下酸性黑ATT的脱色率达87.35%.同时研究了金属离子、酶抑制剂、脱色助剂对酸性黑ATT脱色的影响,结果表明,Co2+、Fe2+、Mn2+、Ca2+、I-对脱色有抑制作用,10mmol·L-1的浓度使脱色率降低了50%以上;Na+、Cu2+在低浓度时对脱色有一定的促进作用;SDS显著抑制漆酶的催化脱色作用,浓度为10mmol·L-1时脱色率降低了68%;HBT是高效的脱色助剂,可显著提高漆酶的脱色效率.     

蜜环菌漆酶对蒽醌类染料的脱色条件优化

利用蜜环菌发酵所得的漆酶,直接对印染工业中常见的两种蒽醌类染料活性艳蓝KN-R和活性艳蓝X-BR进行催化脱色实验,得出了最佳脱色条件.结果表明,活性艳蓝KN-R最适脱色温度为30℃,最适染料浓度为80 mg.L-1,最适酶量为0.25U.mL-1,最适pH值为5,在最优条件下活性艳蓝KN-R最高脱色率达90%以上.活性艳蓝X-BR的最适脱色温度为30℃,最适染料浓度为50 mg.L-1,最适酶量为0.5 U.mL-1,最适pH值为4,在最优条件下活性艳蓝X-BR最高脱色率达70%以上.本研究利用蜜环菌粗漆酶液直接对印染工业中常见蒽醌类染料进行脱色,结果表明蜜环菌粗漆酶液具有良好的脱色效果,蜜环菌漆酶在印染工业染料废水脱色方面具有潜在的应用价值.     

Myrothecium verrucaria NF-08产漆酶条件的响应面法优化及粗酶染料脱色研究

以半知菌属疣孢漆斑菌Myrothecium verrucaria NF-08为供试菌,采用响应面方法优化菌株产漆酶条件,并以其漆酶粗酶进行3种结构类型6种染料的脱色试验,考察了粗酶浓度、温度和p H值对脱色率的影响.两水平析因设计、中心组合设计和验证试验结果表明:葡萄糖浓度为15.4 g·L~(-1)、蛋白胨浓度为43.3 g·L~(-1)和培养基初始p H值为6.68时,漆酶活性达到最大值(25.58±1.60)U·m L~(-1),是单因素试验(16.82 U·m L~(-1))的1.52倍,提高了52.1%.以上述发酵条件获得的M.verrucaria NF-08漆酶粗酶对3种结构类型染料均有脱色效果,排序为偶氮类优于芳甲烷类优于蒽醌类.脱色率随粗酶浓度增大(2~8 U·m L~(-1))、反应温度升高(15~35℃)而上升,随p H值上升(4~10)而下降.粗酶浓度为5 U·m L~(-1),反应温度为25℃,p H值为4时,偶氮类染料铬黑T和芳甲烷类染料碱性品红即可几乎完全脱色,脱色率分别为99.2%和94.6%.     

USTB-05-B基因在大肠杆菌中的表达与纯化研究

采用生物学技术对高效降解线性微囊藻毒素-LR(microcystin-LR,MC-LR)的USTB-05-B酶进行克隆表达及纯化研究。首先利用分子克隆技术得到重组菌pET30a(+)/USTB-05-B/BL21(DE3)。在诱导剂IPTG浓度为0.25 mmol/L、温度30℃和诱导时间4 h条件下,重组菌能大量表达目的蛋白。然后利用亲和层析柱对重组菌破碎后的无细胞提取液(cell free extracts,CE)进行纯化,当咪唑浓度为100 mmol/L时,洗脱下来的蛋白纯度高。最终得到USTB-05-B蛋白纯度为91.1%,浓度为0.205 mg/m L。纯化后的USTB-05-B酶具有较高的活性,能在1 h内将10.8 mg/L的线性MC-LR降解完全。纯化后的目的蛋白为研究USTB-05降解MCs分子机理奠定基础,为有效提高降解MCs速率提供新材料。     

树脂D201上粗漆酶的固定化及对孔雀石绿的脱色

云芝粗漆酶以3种方法,即静电吸附法(D201-Lac-Ⅰ)、先吸附后交联法(D201-Lac-Ⅱ)、戊二醛处理树脂后吸附法(D201-Lac-Ⅲ)固定到大孔阴离子交换树脂D201上.与静电吸附法相比,D201-Lac-Ⅱ的漆酶固载量大幅提升至4.65倍,漆酶比活力却严重受损,只有前者的4.8%;D201-Lac-Ⅲ的漆酶固载量略有下降,为静电吸附法的0.51倍,漆酶比活力却显著提高至静电吸附法的2.99倍.利用电子透射电镜(TEM)可明显观察到D201-Lac-Ⅲ漆酶分子聚合体的阴影.用D201-Lac-Ⅲ对孔雀石绿进行了连续多批次脱色试验,在长达210 h的连续操作中,其脱色效率未观察到有下降趋势,一直稳定在40%～55%之间,D201-Lac-Ⅲ的酶活亦未观察到下降,同等条件下游离的漆酶活力已损失至20%以下,证明D201-Lac-Ⅲ具有显著提升的稳定性和优良的重复利用性.考虑到粗漆酶廉价易得,D201-Lac-Ⅲ在水处理应用中可能大有前景.     

木质素降解菌的分离鉴定及木素过氧化物酶的纯化

采用苯胺蓝脱色法,从活性污泥中分离筛选得到2株具有分泌木质素降解过氧化物酶能力的细菌菌株PKE117和PKE225.根据生理生化反应结果和16SrDNA序列分析,初步确定菌株PKE117为Pseudomanas属的1个新种,PKE225为Pseudomanas thermaerum的1个新菌株.对于菌株PKE117的发酵液依次经DEAE-纤维素32、Sephadex G-75凝胶过滤纯化,木素过氧化物酶的藜芦醇氧化比活力从0.87U/mg提高到204.5U/mg,酶的纯化倍数为235.1,回收率15%.     

Cloning, expression in Escherichia coli, and enzymatic properties of laccase from Aeromonas hydrophila WL-11

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry.It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis.A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized.Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids.The primary structure of the enzyme predicted the structural features characteristic of other laccases,including the conserved regions of four histidine-rich copper-binding sites.The predicted amino acid sequence showed a high homology(more than 60%) with bacterial laccases in the genome and protein databases and the highest degree of similarity(61% identity) was observed with the multicopper oxidase of Klebsiella sp.601.When expressed in Escherichia coli,the recombinant enzyme was overproduced in the cytoplasm as soluble and active form.The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS(2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid) and DMP(2,6-dimethoxyphenol),respectively.The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.     

Potential of plant polyphenol oxidases in the decolorization and removal of textile and non-textile dyes

In this study an effort has been made to use plant polyphenol oxidases; potato （Solanum tuberosum） and brinjal （Solanum melongena）, for the treatment of various important dyes used in textile and other industries. The ammonium sulphate fractionated enzyme preparations were used to treat a number of dyes under various experimental conditions. Majority of the treated dyes were maximally decolorized at pH 3.0. Some of the dyes were quickly decolorized whereas others were marginally decolorized. The initial first hour was sufficient for the maximum decolorization of dyes. The rate of decolorization was quite slow on long treatment of dyes. Enhancement in the dye decolorization was noticed on increasing the concentration of enzymes. The complex mixtures of dyes were treated with both preparations of polyphenol oxidases in the buffers of varying pH values. Potato polyphenol oxidase was significantly more effective in decolorizing the dyes to higher extent as compared to the enzyme obtained from brinjal polyphenol oxidase. Decolorization of dyes and their mixtures, followed by the formation of an insoluble precipitate, which could be easily removed simply by centrifugation.     

Production of laccase by Coriolus versicolor and its application in decolorization of dyestuffs: (Ⅱ) Decolorization of dyes by laccase containing fermentation broth with or without self-immobilized mycelia

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Trametes sp.LS-10C产漆酶发酵培养基优化及其漆酶对偶氮染料的脱色性能

为了提高菌株Trametes sp.LS-10C的漆酶产量并初步研究该酶对偶氮染料的脱色性能,本文通过单因素实验及均匀设计对菌株Trametes sp.LS-10C产漆酶培养基进行了优化,获得最优培养基配方为:葡萄糖14.4 g·L~(-1)、豆渣10.1 g·L~(-1)、麸皮100 g·L~(-1)、NH_4Cl_3g·L~(-1)、KH2PO41.4g·L~(-1)、CuSO_4’5H2O 1 g·L~(-1)、NaCl 1 g·L~(-1)、MgSO_40.8 g·L~(-1)、CaCl_20.5 g·L~(-1).菌株发酵至8 d时漆酶产量为595.15 U·m L~(-1),约是优化前的35.66倍.进一步研究表明,该漆酶-介体系统对酸性红GR、酸性蓝40和酸性铬蓝K等3种偶氮染料脱色10 h后脱色率分别高达96.86%、91.28%和86.31%.相比公开发表的文献,本研究所报道的Trametes sp.LS-10C利用优化培养基发酵产漆酶达到了较高水平,具有酶活力高和发酵时间短等特征,且该漆酶在处理偶氮染料废水脱色领域中具有进一步研发和应用价值.     

Citrobacter sp. LW-3对偶氮染料甲基橙的降解脱色特性研究

从长期受染料污染的土壤中分离出一株能对甲基橙高效降解脱色的柠檬酸盐杆菌Citrobacter sp.LW-3.菌株LW-3在添加了0.5%(g/100 mL)葡萄糖的MSM-1培养基中,16 h使100 mg·L-1甲基橙降解掉90.56%.该菌株对甲基橙降解脱色可在完全好氧条件下,脱色与木质素过氧化物酶、NADH-DCIP原酶、核黄素还原酶有关.菌株LW-3降解甲基橙的适宜温度较广,20~40℃24 h均能使超过80%的甲基橙降解脱色,适宜pH范围为6.0~8.0.通气量对菌株LW-3影响较小,24 h各装液量组(25~200 mL)甲基橙均能降解近90%.同时,菌株LW-3具有较广的染料降解谱,3 d内可使20 mg·L-1另3种偶氮染料、两种三苯甲烷类染料和一种蒽醌类染料很好脱色.菌株LW-3在染料废水的生物处理方面有较强的实际应用价值.     

Production of laccase by Coriolus versicolor and its application in decolorization of dyestuffs: (Ⅱ) Decolorization of dyes by laccase containing fermentation broth with or without self-immobilized mycelia

The capability of decolorization for commercial dyes by Coriolus versicolur fermentation broth containing laccase with or without immobilized mycelium was evaluated.With cell-free fermentation broth containing laccase,high decolorization ratio was achieved for acid orange 7,but not for the other dyes concerned.The immobilized mycelium was proved to be more efficient than the cell-free system.All the four dyestuffs studied were found being decolourized with certain extent by immobilized mycelium.The repeated-batch decolorization was carried out with satisfactory results.The experimental data showed that the continuous decolorization of wastewater from a printing and dyeing industry was possible by using self-immobilized C.Versicolor.     

提高平菇深层培养木质素降解酶活性和对直接湖蓝5B脱色率的探索性研究

以稻草为主要营养基质对平菇进行深层培养,研究了不同浓度(100、200、400、600、800 mg·L-1)直接湖蓝5B对平菇菌丝体产量的影响,以及平菇对不同浓度(100、150、200、250、300、400 mg·L-1)直接湖蓝5B的脱色率.此外,还研究了平菇分别与3种酵母菌(酿酒酵母、产朊假丝酵母和热带假丝酵母)混菌培养及用不同浓度(50、100、200、300、400 mg·L-1)直接湖蓝5B预适应培养的平菇菌种对木质素降解酶活性和直接湖蓝5B脱色率的影响.结果表明:100~400 mg·L-1的直接湖蓝5B均可显著提高了平菇菌丝体产量,而800 mg·L-1的染料对菌丝体的生长有显著抑制作用;染料浓度越高,平菇对其的脱色率越低,发酵时间越长,脱色效果越好.与平菇单菌发酵相比,平菇分别与酿酒酵母和产朊假丝酵母混菌培养可显著提高发酵体系漆酶的活性,平菇分别与所试3种酵母菌混菌培养时锰过氧化物酶活性均有显著增加,与产朊假丝酵母混菌培养时两种酶活性的增加量最大;用50、100、200 mg·L-1直接湖蓝5B预适应培养的菌种接种可显著提高平菇分泌的漆酶的活性,而50mg·L-1和100 mg·L-1的则显著提高锰过氧化物酶的活性.平菇分别与所试3种酵母混菌培养,以及用50 mg·L-1和100 mg·L-1直接湖蓝5B预培养的平菇菌种接种,发酵体系对直接湖蓝5B的脱色效果有显著促进作用,其中,平菇与产朊假丝酵母混菌培养时脱色率最高,达96.3%.通过比较直接湖蓝5B脱色率与漆酶、锰过氧化物酶活性之间的关系可以看出,漆酶、锰过氧化物酶活性与脱色率间基本呈正相关关系.